The gut microbiota are critical to health and development of host animals. This microbial assemblage interacts with a complex host organ that includes the epithelial, immune and nervous tissues. By deriving germ free animals, we can dissect the molecular and cellular mechanisms used by microbes to direct developmental programs in host tissues and that contribute to GI-tract pathologies, such as Inflammatory Bowel Disease and colorectal cancer. My sponsor's lab has demonstrated that the microbiota of the zebrafish, Danio rerio, influences cell-fate specification in the intestinal epithelium. The alterations of intestinal epithelial cell populations in germ free zebrafish are inverse to those found in mutants of the Notch pathway, suggesting that Notch signaling mediates microbiota-directed intestinal development in the both the epithelium and enteric nervous system. Objective: This proposal will explore the hypothesis that Notch signaling mediates microbiota- directed cell-fate specification in the intestinal epithelium and enteric nervous system. In addition, I will identify host genes that modulate interactions with the microbiota during intestinal development using a forward genetic screen and a targeted gene approach. Specific Aims: (1) Determine whether Notch signaling in the gut epithelium is necessary to mediate microbiota-driven cell fate determination and if Notch signaling in the gut epithelium is sufficient to determine epithelial cell fates;(2) Determine whether Notch signaling in the gut epithelium is necessary to mediate microbiota-driven cell fate determination in the enteric nervous system and if Notch signaling in the gut epithelium is sufficient to determine cell fates in the enteric nervous system;and (3) Identify zebrafish genes that contribute to cell fate specification in the gut epithelium in response to the presence of the microbiota. Study Design: I will generate transgenic zebrafish expressing either dominant negative or constitutively active Notch signaling genes under the control of an intestinal epithelium specific promoter. Transgenic zebrafish larvae will be assayed for intestinal secretory and enteric glial cell numbers and molecular markers. To address the interplay between Notch signaling and the microbiota, I will enumerate the intestinal secretory cells and enteric glia in the transgenic lines reared germ free. To identify genes that mediate microbiota specified cell fates, I will perform a chemical mutagenesis followed by early pressure to generate homozygous mutant F2 progeny. Putative mutants with altered intestinal secretory cell numbers will be reared germ free to identify mutants that are specifically deficient in mediating microbiota-directed development. Finally, to test the role of inflammation in microbiota-directed cell fate specification, intestinal secretory cells will be enumerated following the inhibition of either of the innate immune genes myd88 and tnf1R1 by morpholino injection. PUBLIC HEALTH RELEVANCE: The human gut carries a massive number of bacterial cells that contribute to the maintenance of human health, however the molecular processes that underlay the interactions of these bacteria with their animal hosts are not well understood. My proposal aims to use a model fish to determine the mechanisms through which animals respond to their normal bacterial partners. This information will help us understand how these interactions are perturbed in human gut diseases and may lead to novel therapies for the treatment of inflammatory bowel diseases or even intestinal cancers.